The present invention is directed to compounds of the formula 
and pharmaceutically acceptable salts thereof. As used in formula I, and throughout the specification, the symbols have the following meanings:
R1xe2x95x90R2, COR3, CONH2, CONR2R3, COOR2, or SO2R2;
R2=alkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, heterocycloalkylalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl;
R3xe2x95x90H, alkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, heterocycloalkylalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl; 
where n=0,1,2; m=1,2 but both n and m cannot be 2, or 
where i, j=0 or 1 but cannot both be 1, and Y=optionally substituted alkene, alkyne, or any 2 adjacent carbon atoms of a cycloalkyl or cycloheteroalkyl ring of 3-7 atoms;
R4=alkyl with two or more carbon atoms, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, heterocycloalkylalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl, or R9, with the proviso that when R1 is acetyl or propionyl and Y=alkene, then R4 cannot be nitrofuryl or 2-quinolinyl;
R5, R6, R7, R8=independently H, alkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, heterocycloalkylalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl, halo, or hydroxy, alkoxy, amino, NR12R13, thio, or alkylthio, with the proviso that only one such heteroatom group is bonded to any one carbon atom; 
where Zxe2x95x90O, NR14, S;
R10, R11=independently H, alkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, heterocycloalkylalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl, halo, hydroxy, alkoxy, alkylcarbonyloxy, carboxy, alkyloxycarbonyl, amino, NR15R16, carbamoyl, ureido, thio, or alkylthio;
R12, R13, R14, R15, R16=independently H, alkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, heterocycloalkylalkyl, aryl, heteroaryl, arylalkyl, or heteroarylalkyl.
The compounds of formula I are protein kinase inhibitors and are useful in the treatment of proliferative diseases, for example, cancer, inflammation and arthritis. They may also be useful in the treatment of Alzheimer""s disease, and cardiovascular disease.
The present invention provides for compounds of formula I, pharmaceutical compositions employing such compounds, and for methods of using such compounds.
Listed below are definitions of various terms used to describe the compounds of the instant invention. These definitions apply to the terms as they are used throughout the specification (unless they are otherwise limited in specific instances) either individually or as part of a larger group.
It should be noted that any heteroatom with unsatisfied valances is assumed to have the hydrogen atom to satisfy the valances.
Carboxylate anion refers to a negatively charged group xe2x80x94COOxe2x88x92.
The term xe2x80x9calkylxe2x80x9d or xe2x80x9calkxe2x80x9d refers to a monovalent alkane (hydrocarbon) derived radical containing from 1 to 12 carbon atoms unless otherwise defined. An alkyl group is an optionally substituted straight, branched or cyclic saturated hydrocarbon group. When substituted, alkyl groups may be substituted with up to four substituent groups, R as defined, at any available point of attachment. When the alkyl group is said to be substituted with an alkyl group, this is used interchangeably with xe2x80x9cbranched alkyl groupxe2x80x9d. Exemplary unsubstituted such groups include methyl, ethyl, propyl, isopropyl, n-butyl, t-butyl, isobutyl, pentyl, hexyl, isohexyl, heptyl, 4,4-dimethylpentyl, octyl, 2,2,4-trimethylpentyl, nonyl, decyl, undecyl, dodecyl, and the like. Exemplary substituents may include but are not limited to one or more of the following groups: halo (such as F, Cl, Br, I), haloalkyl (such as CCl3 or CF3), alkoxy, alkylthio, hydroxy, carboxy (xe2x80x94COOH), alkyloxycarbonyl (xe2x80x94COOR), alkylcarbonyloxy (xe2x80x94OCOR), amino (xe2x80x94NH2), carbamoyl (xe2x80x94NHCOORxe2x80x94 or xe2x80x94OCONHRxe2x80x94), urea (xe2x80x94NHCONHRxe2x80x94), amidinyl (xe2x80x94CNHNHR or xe2x80x94CNRNH2), or thiol (xe2x80x94SH). Alkyl groups as defined may also comprise one or more carbon to carbon double bonds or one or more carbon to carbon triple bonds.
The term xe2x80x9calkenylxe2x80x9d refers to a hydrocarbon radical straight, branched or cyclic containing from 2 to 12 carbon atoms and at least one carbon to carbon double bond.
The term xe2x80x9calkynylxe2x80x9d refers to a hydrocarbon radical straight, branched or cyclic containing from 2 to 12 carbon atoms and at least one carbon to carbon triple bond.
Cycloalkyl is a specie of alkyl containing from 3 to 15 carbon atoms, without alternating or resonating double bonds between carbon atoms. It may contain from 1 to 4 rings. Exemplary unsubstituted such groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc. Exemplary substituents include one or more of the following groups: halogen, alkyl, alkoxy, alkyl hydroxy, amino, nitro, cyano, thiol and/or alkylthio.
The terms xe2x80x9calkoxyxe2x80x9d or xe2x80x9calkylthioxe2x80x9d, as used herein, denote an alkyl group as described above bonded through an oxygen linkage (xe2x80x94Oxe2x80x94) or a sulfur linkage (xe2x80x94Sxe2x80x94), respectively.
Sulfoxide and sulfone denote groups bonded by xe2x80x94SOxe2x80x94 or xe2x80x94SO2xe2x80x94 linkages, respectively.
The term xe2x80x9calkyloxycarbonylxe2x80x9d, as used herein, denotes an alkoxy group bonded through a carbonyl group. An alkoxycarbonyl radical is represented by the formula: xe2x80x94C(O)OR, where the R group is a straight or branched C1-6 alkyl group.
The term xe2x80x9calkylcarbonylxe2x80x9d refers to an alkyl group bonded through a carbonyl group.
The term xe2x80x9calkylcarbonyloxyxe2x80x9d, as used herein, denotes an alkylcarbonyl group which is bonded through an oxygen linkage.
The term xe2x80x9carylalkylxe2x80x9d, as used herein, denotes an aromatic ring bonded to an alkyl group as described above.
The term xe2x80x9carylxe2x80x9d refers to monocyclic or bicyclic aromatic rings, e.g. phenyl, substituted phenyl and the like, as well as groups which are fused, e.g., napthyl, phenanthrenyl and the like. An aryl group thus contains at least one ring having at least 6 atoms, with up to five such rings being present, containing up to 22 atoms therein, with alternating (resonating) double bonds between adjacent carbon atoms or suitable heteroatoms. Aryl groups may optionally be substituted with one or more groups including, but not limited to halogen, alkyl, alkoxy, hydroxy, carboxy, carbamoyl, alkyloxycarbonyl, nitro, trifluoromethyl, amino, cycloalkyl, cyano, alkyl S(O)m (m=, 0,1, 2), or thiol.
The term xe2x80x9cheteroarylxe2x80x9d refers to a monocyclic aromatic hydrocarbon group having 5 or 6 ring atoms, or a bicyclic aromatic group having 8 to 10 atoms, containing at least one heteroatom, O, S, or N, in which a carbon or nitrogen atom is the point of attachment, and in which one or two additional carbon atoms is optionally replaced by a heteroatom selected from O or S, and in which from 1 to 3 additional carbon atoms are optionally replaced by nitrogen heteroatoms, said heteroaryl group being optionally substituted as described herein. Exemplary heteroaryl groups include the following: thienyl, furyl, pyrrolyl, pyridinyl, imidazolyl, pyrrolidinyl, piperidinyl, thiazolyl, oxazolyl, triazolyl, pyrazolyl, isoxazolyl, isothiazolyl, pyrazinyl, tetrazolyl, pyridazinyl, pyrimidinal, triazinylazepinyl, indolyl, isoindolyl, quinolinyl, isoquinolinyl, benzothiazolyl, benzoxazolyl, benzimidazolyl, benzoxadiazolyl, benzofurazanyl and tetrahydropyranyl. Exemplary substituents include one or more of the following: halogen, alkyl, alkoxy, hydroxy, carboxy, carbamoyl, alkyloxycarbonyl, trifluoromethyl, cycloalkyl, nitro, cyano, amino, alkylS(O)m (m=, 0,1, 2), or thiol.
The term xe2x80x9cheteroaryliumxe2x80x9d refers to heteroaryl groups bearing a quaternary nitrogen atom and thus a positive charge.
The term xe2x80x9cheterocycloalkylxe2x80x9d refers to a cycloalkyl group (nonaromatic) in which one of the carbon atoms in the ring is replaced by a heteroatom selected from O, S or N, and in which up to three additional carbon atoms may be replaced by said heteroatoms.
The term xe2x80x9cquaternary nitrogenxe2x80x9d refers to a tetravalent positively charged nitrogen atom including, e.g. the positively charged nitrogen in a tetraalkylammonium group (e.g. tetramethylammonium, N-methylpyridinium), the positively charged nitrogen in protonated ammonium species (e.g. trimethylhydroammonium, N-hydropyridinium), the positively charged nitrogen in amine N-oxides (e.g. N-methyl-morpholine-N-oxide, pyridine-N-oxide), and the positively charged nitrogen in an N-amino-ammonium group (e.g. N-aminopyridinium).
The term xe2x80x9cheteroatomxe2x80x9d means O, S or N, selected on an independent basis.
The term xe2x80x9chalogenxe2x80x9d or xe2x80x9chaloxe2x80x9d refers to chlorine, bromine, fluorine or iodine.
When a functional group is termed xe2x80x9cprotectedxe2x80x9d, this means that the group is in modified form to preclude undesired side reactions at the protected site. Suitable protecting groups for the compounds of the present invention will be recognized from the present application taking into account the level of skill in the art, and with reference to standard textbooks, such as Greene, T. W. et al., Protective Groups in Organic Synthesis, Wiley, N.Y. (1991).
Suitable examples of salts of the compounds according to the invention with inorganic or organic acids are hydrochloride, hydrobromide, sulfate, phosphate. Salts which are unsuitable for pharmaceutical uses but which can be employed, for example, for the isolation or purification of free compounds I or their pharmaceutically acceptable salts, are also included.
All stereoisomers of the compounds of the instant invention are contemplated, either in admixture or in pure or substantially pure form. The definition of the compounds according to the invention embraces all possible stereoisomers and their mixtures. It very particularly embraces the racemic forms and the isolated optical isomers having the specified activity. The racemic forms can be resolved by physical methods, such as, for example, fractional crystallization, separation or crystallization of diastereomeric derivatives or separation by chiral column chromatography. The individual optical isomers can be obtained from the racemates by conventional methods, such as, for example, salt formation with an optically active acid followed by crystallization.
All configurational isomers of compounds of the present invention are contemplated, either in admixture or in pure or substantially pure form. The definition of compounds of the present invention very particularly embraces both cis (Z) and trans (E) alkene isomers, as well as cis and trans isomers of cycloalkyl or heterocycloalkyl rings.
It should be understood that solvates (e.g. hydrates) of the compounds of formula I are also within the scope of the present invention. Methods of salvation are generally known in the art. Accordingly, the compounds of the instant invention may be in the free or hydrate form, and may be obtained by methods exemplified by the following schemes.
The synthesis of compounds of formula I can proceed through the known aldehyde of formula II (Scheme 1) which was prepared according to the procedures set forth in II Farmaco 44, 1011, (1989) and the references therein. Treatment of II with either (R2CO)2O or R1xe2x80x94L, where L is a leaving group such as a halogen or sulfonate ester, yields compounds of formula III. Condensation of formula III with phosphorus-stabilized anions such as the phosphonate of formula IV or a Wittig reagent in the presence of base yields compounds of formula V (that is, compounds of formula I where A contains an alkene present as either the cis or trans isomer). Alternatively, compounds of formula V may be prepared by first reacting formula II with the phosphonate of formula IV or a Wittig reagent in the presence of base, and then treating the resulting product with (R2CO)2O or R1xe2x80x94L. 
Compounds of formula V may be converted into other compounds of formula I as shown in Scheme 2. For example, treatment of compounds of formula V with agents such as H2 on Pd/C yields the saturated compounds of formula VI (which is a compound of formula I. Alternatively compounds of formula V may be epoxidized with agents such as dimethyldioxirane or m-chloroperbenzoic acid to yield epoxides of formula VII (which are compounds of formula I where Y=the carbon atoms of oxirane). Cyclopropanation of the olefin with agents such as ZnCuCH2 or diazomethane may yield cyclopropanes of formula VIII (which are compounds of formula I where Y=cyclopropane). 
Aldehydes of formula III may also be converted into compounds of formula I which have R7 or R8 groups containing oxygen (Scheme 3). For example, addition of organometallic reagents of formula R*-M, where R*xe2x95x90R4(R5R6C)ixe2x80x94 or R4(R5R6C)Ixe2x80x94Yxe2x80x94 and M=a metal, would yield compounds of formula IX (that is, compounds of formula I where R7=hydroxy and R8xe2x95x90H). Alkylation of the hydroxyl group in compounds of formula IX using Wxe2x80x94L, where W=alkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, heterocycloalkylalkyl, aryl, heteroaryl, arylalkyl, or heteroarylalkyl, and L is a leaving group such as a halogen or sulfonate ester, would yield ethers of formula X (that is, compounds of formula I with R7=alkoxy). 
Scheme 4 outlines a procedure that may be used for the solid phase synthesis of compounds of formula I. A benzyl chloride resin, such as that depicted by formula XII, may be alkylated by an aminothiazole of formula III (where R1xe2x95x90CF3CO) to give a compound of formula XII. Coupling with phosphorus stabilized anions such as compounds of formula IV will yield alkenes of formula XIII which may be deprotected by a reducing agent such as sodium borohydride, or a base such as sodium hydroxide, to give amines of formula XIV. The amines of formula XIV may react with R1xe2x80x94L or (R2CO)2O to give compounds of formula XV, which may be cleaved from the resin with trifluoroacetic acid to give compounds of formula V (which are compounds of formula I where Y is an alkene). Compounds of formula IX or X may also be synthesized on solid phase using analogous chemistry to that shown in Scheme 3 by starting with aldehyde XII. 
Compounds of formula I wherein R4xe2x95x90R9 may be synthesized from aldehydes of formula III (Scheme 5). These aldehydes may be reduced with agents such as sodium borohydride to give alcohols of formula XVI which may be converted into a compound of formula XVII, where L is a leaving group such as a halogen or sulfonate ester, by treatment with agents such as p-toluenesulfonyl chloride and base or thionyl chloride. The anion of dialkylmalonate esters of formula XVIII may be alkylated by compounds of formula XVII to form diesters of formula XIX, where W=alkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, heterocycloalkylalkyl, aryl, heteroaryl, arylalkyl, or heteroarylalkyl. These diesters may be saponified and decarboxylated to form acids of formula XX which may be coupled with amines of formula XXI to give amides of formula XXII. These amides may be cyclized upon exposure to dehydrating agents such as POCl3 to form compounds of formula VI which are compounds of formula I where Zxe2x95x90O. 
Compounds of formula I wherein R4=R9 and Y=alkynyl or Z-alkenyl may be prepared from halomethyl oxazoles such as XXIII (Scheme 6). Displacement of the chlorine to give the acetate XXIV, followed by basic hydrolysis and oxidation provides a 2-oxazolyl aldehyde XXVI. The aldehyde may be treated with a reagent such as carbon tetrabromide and triphenylphosphine to give a dibromo olefin XXVII. Elimination of HBr by strong base, followed by lithiation and quenching the acetylenic anion with tributyltin chloride gives an acetylenic stannane XXVIII, which may be coupled with a 2-iodo aminothiazole XXIX, to give XXX, which is a compound of formula I wherein R4xe2x95x90R9 and Y is alkynyl. The acetylenic compounds of formula XXX may be hydrogenated to provide cis olefins XXXI and XXXIII, which are compounds of formula I wherein R4xe2x95x90R9 and Y is Z-alkenyl. 
Compounds of formula I wherein R1=R2 may be prepared by the methods shown in Scheme 7. 5-Formyl-2-aminothiazole II is reacted with a nitrosating agent such as tBuONO and CuBr2 to form the 2-bromo derivative XXXIV. Following procedures as described in Scheme I, the aldehyde is reacted with a phosphonate of formula IV or a Wittig reagent in the presence of base to provide an olefin of formula XXXV. Reaction of the 2-bromo olefin of formula XXXV with a compound of formula R2NH2 in the presence of a base such as sodium hydride gives compounds of formula, which is a compound of formula I wherein R1=R2 and Y is alkenyl. 
Alternatively, compounds of formula XXXVI where R1xe2x95x90R2 and Y is alkenyl may be prepared according to Scheme 8. The amino group of compound II may be protected with a reagent such as di-t-butyl dicarbonate to give XXXVII, followed by reaction with a phosphonate of formula IV or a Wittig reagent in the presence of base such as an alkoxide or sodium hydride to give a compound of formula XXXVIII. Treatment of XXXVIII with R2L where L is a leaving group such as halo or sulfonate, in the presence of base, followed by removal of the protecting group gives a compound of formula XXXVI, which is a compound of formula I where R1xe2x95x90R2 and Y is alkenyl. 
Compounds of formula R4CH2P(O)(OEt)2 may be prepared from compounds of formula R4CH2L, where L is a leaving group such as halogen or sulfonate ester, by heating with triethylphosphite. Compounds of formula R9-L, where Zxe2x95x90O, may be prepared from LCH2CN and R11C(N2)COR10, according to part E of Example 2.
The starting compounds of Schemes 1-7 are commercially available or may be prepared by methods known to one of ordinary skill in the art.
All compounds of formula I may be prepared by modification of the procedures described herein.
Preferred compounds of formula I are those where:
R1xe2x95x90R2, COR3, or CONR2R3;
R2xe2x95x90alkyl, aryl, or heteroaryl;
R3xe2x95x90H, alkyl, aryl, heteroaryl, arylalkyl, or heteroarylalkyl; 
where n=0, 1, 2; m=1, 2, or 
where i, j=0 or 1 but cannot both be 1, and Y=optionally substituted alkene, alkyne, or any two adjacent carbon atoms of a cycloalkyl ring;
R4=alkyl with two or more carbon atoms, aryl, heteroaryl, or R9;
R5, R6, R7, R8=independently H, or alkyl; 
where Zxe2x95x90O;
R10, R11=independently H, or alkyl.
The compounds according to the invention have pharmacological properties; in particular, the compounds of formula I are inhibitors of protein kinases such as the cyclin dependent kinases (cdks), for example, cdc2 (cdk1), cdk2, and cdk4. The novel compounds of formula I are expected to be useful in the therapy of proliferative diseases such as cancer, inflammation, arthritis, Alzheimer""s disease and cardiovascular disease. These compounds may also be useful in the treatment of topical and systemic fungal infections.
More specifically, the compounds of formula I are useful in the treatment of a variety of cancers, including (but not limited to) the following:
carcinoma, including that of the bladder, breast, colon, kidney, liver, lung, ovary, pancreas, stomach, cervix, thyroid, prostate, and skin;
hematopoietic tumors of lymphoid lineage, including acute lymphocytic leukemia, B-cell lymphoma, and Burkett""s lymphoma;
hematopoietic tumors of myeloid lineage, including acute and chronic myelogenous leukemias and promyelocytic leukemia;
tumors of mesenchymal origin, including fibrosarcoma and rhabdomyosarcoma; and
other tumors, including melanoma, seminoma, teratocarcinoma, osteosarcoma, neuroblastoma and glioma.
Due to the key role of cdks in the regulation of cellular proliferation in general, inhibitors could act as reversible cytostatic agents which may be useful in the treatment of any disease process which features abnormal cellular proliferation, e.g., neuro-fibromatosis, atherosclerosis, pulmonary fibrosis, arthritis, psoriasis, glomerulonephritis, restenosis following angioplasty or vascular surgery, hypertrophic scar formation, inflammatory bowel disease, transplantation rejection, angiogenesis, and endotoxic shock.
Compounds of formula I may also be useful in the treatment of Alzheimer""s disease, as suggested by the recent finding that cdk5 is involved in the phosphorylation of tau protein (J. Biochem, 117, 741-749 (1995)).
Compounds of formula I may also act as inhibitors of other protein kinases, e.g., protein kinase C, her2, rafl, MEK1, MAP kinase, EGF receptor, PDGF receptor, IGF receptor, Pl3 kinase, wee1 kinase, Src, Abl, and thus be effective in the treatment of diseases associated with other protein kinases.
The compounds of this invention may also be useful in combination with known anti-cancer treatments such as radiation therapy or with cytostatic and cytotoxic agents, such as for example, but not limited to, DNA interactive agents, such as cisplatin or doxorubicin; inhibitors of farnesyl protein transferase, such as those described in pending U.S. application Ser. No. 08/802,239 which was filed on Feb. 20, 1997; topoisomerase II inhibitors, such as etoposide; topoisomerase I inhibitors, such as CPT-11 or topotecan; tubulin stabilizing agents, such as paclitaxel, docetaxel or the epothilones; hormonal agents, such as tamoxifen; thymidilate synthase inhibitors, such as 5-fluorouracil; and antimetabolites, such as methoxtrexate; antiangiogenic agents, such as angiostatin; and kinase inhibitors, such as her2 specific antibodies.
If formulated as a fixed dose, such combination products employ the compounds of this invention within the dosage range described below and the other pharmaceutically active agent within its approved dosage range. For example, the cdc2 inhibitor olomucine has been found to act synergistically with known cytotoxic agents in inducing apoptosis (J. Cell Sci., 108, 2897 (1995)). Compounds of formula I may be used sequentially with known anti-cancer or cytotoxic agents when a combination formulation is inappropriate.
cdc2/cyclin B1 Kinase Assay
cdc2/cyclin B1 kinase activity was determined by monitoring the incorporation of 32P into histone HI. The reaction consisted of 50 ng baculovirus expressed GST-cdc2, 75 ng baculovirus expressed GST-cyclin B1, 1 xcexcg histone HI (Boehringer Mannheim), 0.2 xcexcCi of 32P xcex3-ATP and 25 xcexcM ATP in kinase buffer (50 mM Tris, pH 8.0, 10 mM MgCl2, 1 mM EGTA, 0.5 mM DWF). The reaction was incubated at 30xc2x0 C. for 30 minutes and then stopped by the addition of cold trichloroacetic acid (TCA) to a final concentration of 15% and incubated on ice for 20 minutes. The reaction was harvested onto GF/C unifilter plates (Packard) using a Packard Filtermate Universal harvester, and the filters were counted on a Packard TopCount 96-well liquid scintillation counter (Marshak, D. R., Vanderberg, M. T., Bae, Y. S., Yu, I. J., J. of Cellular Biochemistry, 45, 391-400 (1991), incorporated by reference herein).
cdk2/cyclin E Kinase Assay
cdk2/cyclin E kinase activity was determined by monitoring the incorporation of 32P into the retinoblastoma protein. The reaction consisted of 2.5 ng baculovirus expressed GST-cdk2/cyclin E, 500 ng bacterially produced GST-retinoblastoma protein (aa 776-928), 0.2 xcexcCi 32P xcex3-ATP and 25 xcexcM ATP in kinase buffer (50 mM Hepes, pH 8.0, 10 mM MgCl2, 5 mM EGTA, 2 mM DTT). The reaction was incubated at 30xc2x0 C. for 30 minutes and then stopped by the addition of cold trichloroacetic acid (TCA) to a final concentration of 15% and incubated on ice for 20 minutes. The reaction was harvested onto GF/C unifilter plates (Packard) using a Packard Filtermate Universal harvester, and the filters were counted on a Packard TopCount 96-well liquid scintillation counter.
cdk 4/cyclin D1 Kinase Activity
cdk4/cyclin D1 kinase activity was determined by monitoring the incorporation of 32P in to the retinoblastoma protein. The reaction consisted of 165 ng baculovirus expressed as GST-cdk4, 282 ng bacterially expressed as S-tag cyclin D1, 500 ng bacterially produced GST-retinoblastoma protein (aa 776-928), 0.2 xcexcCi 32P xcex3-ATP and 25 xcexcM ATP in kinase buffer (50 mM Hepes, pH 8.0, 10 mM MgCl2, 5 mM EGTA, 2 mM DTT). The reaction was incubated at 30xc2x0 C. for 1 hour and then stopped by the addition of cold trichloroacetic acid (TCA) to a final concentration of 15% and incubated on ice for 20 minutes. The reaction was harvested onto GF/C unifilter plates (Packard) using a Packard Filtermate Universal harvester, and the filters were counted on a Packard TopCount 96-well liquid scintillation counter (Coleman, K. G., Wautlet, B. S., Morissey, D, Mulheron, J. G., Sedman, S., Brinkley, P., Price, S., Wedster, K. R. (1997) Identification of CDK4 Sequences involved in cyclin D, and p16 binding. J. Biol. Chem. 272,30:18869-18874, incorporated by reference herein).
The following examples and preparations describe the manner and process of making and using the invention and are illustrative rather than limiting. It should be understood that there may be other embodiments which fall within the spirit and scope of the invention as defined by the claims appended hereto.